The field of this invention is the area of protein glycosylation, specifically the area of the particular enzyme, UDP N-acetylglucosaminyltransferase V, involved in the expression of the β(1,6) branch structure found in tri- and tetra-antennary NBlinked oligosaccharides. The field relates to the amino acid sequences of rat, human and hamster GlcNAc T-V proteins, genes encoding active enzyme and cell lines genetically engineered to express a nucleotide sequence encoding active enzyme.
UDP-N-acetylglucosamine:α-6-D-mannoside β-1,6-N-acetylglucosaminyltransferase V (EC 2.4.1.155) is the Golgi enzyme responsible for the synthesis of the β(1,6) branch structure of tri- and tetra-antennary Blinked oligosaccharides. For brevity, this enzyme is abbreviated GlcNAc T-V herein. GlcNAc T-V activity has been found in many tissues and cell types. One GlcNAc T-V protein, termed GlcNAc T-Va herein, has been purified (Shoreibah et al. (1992) J. Biol. Chem. 262: 2920-2927, and the cDNA has been isolated and sequenced (Shoreibah et al. (1993) J. Biol. Chem. 268:15381-15385, U.S. Pat. No. 5,602,003 and No. 6,015,701). GlcNAc T-Va is determined by a gene on chromosome 2.
Altered glycosylation of membrane glycoproteins and glycolipids is observed in mammalian cells transformed with diverse tumor viruses, carcinogens, or transfection with certain oncogenes. In some cases, there is a quantitative increase in a particular substituent, e.g., sialylation. In other instances, there is the reappearance of an oligosaccharide structure in the tumor which is normally only found in fetal tissue; for instance, certain Lewis histo-blood group antigens have been detected in adenocarcinomas.
Qualitative differences in oligosaccharides may also be observed in certain transformed cells. BHK fibroblasts transformed with polyoma virus or with Rous sarcoma virus display more highly branched complex N-linked oligosaccharides than do the corresponding normal cells. The expression of the β-1,6 branch structure (-[GlcNAc-β(1,6)Man-α(1,6)Man]-) found in tri- and tetra-antennary NBlinked oligosaccharides is increased in the transformed cells. This has been correlated with a 2 to 3-fold increase in the specific activity of GlcNAc T-V. Transformation of murine cells with polyoma viruses, adenovirus, tumorigenic DNA and either the ras or the her-2/new oncogenes also resulted in increased GlcNAc T-V activity. By contrast, several other glycosyl transferases involved in N-linked glycosylation are unchanged in the transformed cells. The mechanism for the increased specific activity of GlcNAc T-V in transformed cells is not known.
The increase in the β(1,6) branching of the cell surface-bound oligosaccharides has been associated, at least in some cases, with capacity for metastasis. Increased levels of β-1,6 branching over the level in normal tissue have been observed for some human breast tumor tissues.
Certain mammalian glycosyl transferases from the N-linked glycosylation pathway have been purified and characterized. The enzymatic machinery for the glycosylation of proteins in mammalian cells is generally located in the membranes of the Golgi apparatus. α(1,3) mannoside β(1,2) UDP-N-acetylglucosaminyl transferase I (GlcNAc T-I) (EC 2.4.1 101) and UDP-N-acetyl-glucosaminyl transferase II (GlcNAc T-II) (EC 2.4.1.143) have been purified from rabbit liver and rat liver, respectively. GlcNAc T-I has been purified 7000-fold from a Triton X-100 extract of rabbit liver acetone powder by two rounds of affinity chromatography over UDP-hexanolamine agarose, in the first round by elution with NaCl, and in the second round by elution with UDP (Oppenheimer and Hill (1981) J. Biol. Chem. 256: 799-804). GlcNAc T-I (UDP-N-acetylglucosaminyl:α-D-mannoside β(1,2) Bacetylglucosaminyltransferase II) was purified 60,000-fold from rat liver by Triton X-100 extraction of rat liver membranes, followed by chromatography over carboxymethyl-cellulose, hydroxylapatite, and sequential elutions using NaCl, UDP-GlcNAc and EDTA from 5-mercuri-UDP-GlcNAc-thiopropyl-SEPHAROSE, Affi-Gel (Bio-Rad Laboratories, Richmond, Calif.) blue affinity chromatography and finally UDP-GlcNAc-SEPHAROSE (Bendiak and Schachter (1987) J. Biol. Chem. 262: 5775-5783).
The cDNA encoding a rat liver Golgi sialyl transferase (β-galactoside α(2,6)-sialyl transferase (EC 2.4.99.1) has been cloned and sequenced (Weinstein et al. (1987) J. Biol. Chem. 262: 17735-17743). The corresponding enzyme has been purified 23,000-fold from Triton CF-54 extracts of rat liver membranes by three rounds of affinity chromatography over CDP-hexanolamine-agarose (Weinstein et al. (1982) J. Biol. Chem. 257: 13835-13844). Soluble recombinant glycosyl transferases are described in U.S. Pat. No. 5,032,519, issued Jul. 16, 1991, incorporated by reference herein.
There is a need in the art for enzymes which function in the glycosylation of proteins or in the remodeling of the glycosylation of proteins, especially to improve the glycosylation status of recombinant proteins.